ABSTRACT Lyme disease is the most commonly reported vector-borne disease in North America, with nearly 30,000 confirmed cases and > 8,500 probable cases reported to the CDC in 2009. The etiologic agent, B. burgdorferi is transmitted to humans via Ixodes ticks and spreads hematogenously to cause a systemic disease. The protean nature of this disease lends to difficult clinical diagnosis, so laboratory testing needs to be reliable. An improved diagnostic test for Lyme disease must detect infection: a) early (within 2 weeks of tick bite); b) across multiple B. burgdorferi genospecies, and c) in the different phases of disease. The ability of a diagnostic test to distinguish persistent infection from clinical cure would be an asset as well. New Luminex(R) technology incorporating monospecific antibodies bound to fluorometric beads has allowed for the quantification of multiple proteins within a single sample. We aim to reverse the process by coupling multiple to the beads for use in the detection of specific antibodies for the diagnosis of Lyme disease. Along with the C6 peptide, the most successful standalone diagnostic antigen for Lyme disease, we have carefully selected four additional antigens (DbpA, OspA, OspC and OppA-2) to incorporate into the initial testing of this platform. Each is broadly immunogenic, but elicits different responses over the course of infection. The overall goal is to produce a single diagnostic test for Lyme disease that is sensitive, specific and detects infection with multiple variants at all stages of disease. The objective of the proposed work is to create a platform test, based on Luminex(R) technology and our knowledge of antibody responses to B. burgdorferi throughout infection, which will overcome the deficiencies of current tests. The central hypothesis is that the combination of select antigens that elicit distinctive host antibody response patterns into a fluorometric bead-based quantitative assay will provide assay sensitivity and specificity exceeding that of current Lyme diagnostics. We propose the following aims (Phase I): (1) Proof of concept: to combine antigens into bead arrays for testing and optimization of sensitivity using monoclonal antibodies and immune serum. The working hypothesis for this aim is that monoclonal antibodies can be used to produce standard curves to determine levels of specific antibody in a clinical sample and to define the dynamic range of detection for each antigen; (2) Proof of feasibility for human use: to test assay specificity compared to standard ELISA using serum from multiple sources, including Lyme disease patients at different phases of disease (early, disseminated, or late- disseminated) versus healthy control patient serum. The working hypothesis for this aim is that by coupling to beads only 5 carefully-selected B. burgdorferi-specific purified antigens, the sensitivity of specific antibody detection will be enhanced without compromising specificity. Following the completion of these aims, in Phase II the diagnostic assay will be optimized, streamlined and tested against standard diagnostic methods currently employed for the diagnosis of Lyme disease.